The solid phase synthesis of peptides is a complex process which involves stepwise additions of aminoterminus-blocked amino acids to a peptide chain, the carboxyl terminus of which is attached to a solid support. Solid phase peptide synthesis (SPPS) typically begins with covalent attachment of the carboxyl end of a first alpha-amine protected amino acid through an organic linker to an insoluble resin synthesis bead. The general synthesis cycle then consists of deprotection of the alpha-amine group of the last amino acid, washing and, if necessary, neutralization, followed by reaction with a carboxyl-activated form of the next alpha-amine protected amino acid to be added. Each successive amino acid is attached to the terminal nitrogen by the carbonyl carbon of the carboxylic acid group.
Most present peptide synthesizers perform coupling with carbodi-imide reagents (e.g., with dicyclohexyl carbodi-imide (DCCI), or with diisopropylcarbodiimide (DIPCDI)). The synthesis is carried out in a reaction vessel which includes a synthesis resin therein.
After the coupling reaction is complete, the protected amino acid which is coupled through its carboxylic acid group to the synthesis resin is deblocked with a deblocking agent, such as trifluoroacetic acid (TFA) or piperidine, washed (when TFA is used) with a base and the next activated amino acid residue is added to the reaction vessel. Upon obtaining the desired peptide sequence, the peptide is cleaved from the synthesis support, generally either with anhydrous hydrogen fluoride (HF) or with TFA.
There are basically two ways of performing coupling in automated SPPS: with activated amino acid derivatives or with free amino acid derivatives which are activated prior to coupling. One advantage to using activated derivatives, such as active esters, is that these, if they are crystalline, can be highly pure and give clean couplings. A disadvantage to active esters is that if they are highly reactive, then they will be unstable in solution, and in long term storage in the solid state. Therefore, they must be stored as powders and dissolved prior to coupling.
Pentafluorophenyl (PFP) active esters are currently used in solid phase peptide synthesis and in solution synthesis. These derivatives suffer from the following disadvantages: they are made from pentafluorophenol, a highly toxic, costly and hygroscopic substance. Treatment of wastes is a problem. It is believed that PFP may contain dangerous, dioxin-like materials or give rise to such materials on storage; the pentafluorophenol liberated in this reaction is highly acidic, and protonates amino groups on the resin slowing the final stages of coupling. Since this would be an equilibrium process, protonation by this mild acid could not prevent the coupling from going to completion, but might slow the process; two PFP esters, serine (Ser) and threonine (Thr), are noncrystalline and so for these, alternative crystalline esters are used; and it is believed that the solution stability of PFP esters is insufficient. Thus, viable alternatives for the use of PFP-active esters are needed.
Available systems for solid phase peptide synthesis are described, for example, in U.S. Pat. Nos. 4,362,699, 4,531,258, 3,647,390 and 3,557,077. Ideally, the system should include a method for monitoring the completeness of each amino acid addition before the next amino acid is added to the peptide chain. Such a system would ensure the best possible yields of peptides. Some available methods for monitoring the amino acid synthesis reaction are described by G. Barany and B. Merrifield in "Selected Methods for Monitoring a Solid-Phase Peptide Synthesis", in The Peptides, Vol. 2, pp. 150-154 (1979).
Of the various methods for monitoring solid phase peptide synthesis, the most efficient and sensitive methods available to date to determine the presence of unreacted, or free, amino acids by detecting the amino groups. These methods genera.
Of the various methods for monitoring solid phase peptide synthesis, the most efficient and sensitive methods available to date to determine the presence of unreacted, or free, amino acids by detecting the amino groups. These methods generally involve stopping the reaction, and titrating the resin-bound amino groups with reagents which are reactive with the amino groups, and show a detectable response (e.g., colorimetric or potentiometric). These titration methods generally utilize acids, such as perchloric acid or picric acid, or halogen compounds which form halide salts, such as pyridine hydrochloride. These methods have several drawbacks, including the necessity of stopping the coupling reaction, destruction of some of the amino groups and reactivity with the peptide chain itself.